The present invention concerns to molecular biology and biotechnology, namely to the methods and devices for synthesis of polypeptides in cell-free translation system.
Several methods of polypeptide synthesis in cell-free translation system are known. For elimination of restrictions connected with a lower output of target polypeptides and short-term operation of cell-free translation systems a method was suggested which is widely used now (Spirin et al., 1988). This method is based on the principle of continuous removal from a reaction mixture of reaction products and continuous restoration of the initial concentration of low molecular weight components during synthesis. This method underlies several inventions connected with its improvement for increasing the synthesized product output (Alkahov et al., 1991; Baranov et al., 1993; Alakhov et al., 1995).
By the input of feeding solutions and removal of products of synthesis the known methods can be divided as follows: (a) methods in which dialysis is used to add feed solution components to the reaction mixture and to remove low molecular weight components from the reaction mixture through the dialysis membrane or to simultaneously remove low and high molecular weight components from the reaction mixture; (b) methods in which continuous ultrafiltration is used for a simultaneous removing of low and high molecular weight components of products through the membrane and a simultaneous input of feeding solutions directly into the reaction mixture volume or through the membrane; c) methods in which periodic input of a feed solution into the reaction mixture and subsequent removing of low and high molecular weight components through the membrane are used. Input and output of the flows is realized by changing the direction of liquid flows at the exposure of consecutive creation of pulses of positive or negative pressure.
The method (Mozayeny, 1995) is known in which the removal of products with large molecular weight is improved by increasing the area of a ultrafiltration membrane in relation to the reaction mixture volume. One of the main disadvantages of the given invention is that during removal of high molecular weight components through the large area of the membrane with the pore size of 70 kD to 100 kD, together with the final product useful working components of molecular weight up to 100 kD are lost. This is a limiting factor for the operating time of the cell-free system. The larger is the membrane area, the greater is the amount of high molecular components of the cell-free system washed-off from the reactor at a high flow rate. Another disadvantage is the necessity to use an external loop for creation of a tangential flow of the reaction mixture along the membrane surface. During passage of the reaction mixture via liquid communications three factors influence the work of the cell-free systems: (1) when the reaction mixture passes via the loop the feed solution is not added in the part of external volume of the reaction mixture, (2) low weight products which inhibit the cell-free system are not removed from the external volume, (3) the liquid communications and pumps are not thermostable and the reaction mixture changes its temperature depending on the environment. This leads to irreproducibility of results and limits the life time of the cell-free system.
The method in which authors offer to apply repeated pulse for input of the feeding solution in the reactor and removal of low and high molecular weight products of synthesis from the reactor via a membrane is known (Fischer et al., 1990). This is realized by changing the direction of the flow through the membrane. One of the main disadvantages of the given invention is that low molecular weight components of synthesis which inhibit operation of the system are not removed from the reactor during a long period. The time during which the feed solution passes repeatedly via the membrane is equal to the period when a total volume of feed solution passage via the membrane is equal to the complete volume of the reaction mixture. For this purpose N cycles are formed to create positive and negative pressure. Due to the pressure modulation the inhibiting products come back in the reactor together with a regular portion of the feed solution. Another disadvantage of this method is that upon formation of N cycles high molecular weight components of the cell-free system required for prolonged synthesis are intensively washed off the reaction mixture. Thus, repeated returning into the reactor of low molecular weight components inhibits operation of the system and removing from the reactor of high molecular weight components providing effective synthesis impose restrictions on operation of system.
The method (Choi. 1997) is known by which synthesis of polypeptides is carried out with removal of a target product in a dialysis mode of operation. For this purpose a membrane divides the reactor in two parts. The reaction mixture is placed on one side of the membrane and the feed solution on the other side. The reaction mixture is fastly circulating along the membrane surface in tangential direction. A disadvantage of the method is that due to a large pore size components of the system are removed together with the target products. Moreover, in spite of the fact that the dialysis process is quite effective because of the large pore size, its extent is not enough for operation of highly efficient cell-free systems.
The method (Alakhov et al., 1991) is known in which amino acids, ATP, GTP in an aqueous buffer are added to reactor during functioning of the system and low weight components such as AMP, GDP, Pi formed during synthesis and inhibit the system are removed through a membrane. To provide a more economical operation of the system, low molecular weight products are regenerated and come back into the reactor via the membrane. However from the description and the given figure it is not quite clear how low and high molecular weight components of the synthesis are removed from the reactor and in what way the buffer solution is regenerated after removal of the polypeptide. Taking into account the description of examples, low and high molecular weight components are removed from the reactor via the ultrafiltration membrane. The use of an ultrafiltration membrane is described in a number of publications (Spirin et al., 1988; Takanori et al., 1991; Spirin, 1992; Erdmann et al., 1994). A disadvantage of this method is the use of large sizes of the membrane cutoff. In this case high molecular weight components of systems necessary for synthesis are removed from the reactor together with target products. The volume of low molecular weight components is equal to that of the removed components which results in fast closing of the ultrafiltration membrane pores.
Methods of adding feed solution to the reaction zone and removing from it of reaction products for different types of membrane reactors are known in which the reaction zone is placed between two membranes (Matson et al., 1988; Wrasidlo et al., 1990; Dziewulski et al., 1992).
The method described in the patent (Alakhov et al., 1995) is the prototype of the method proposed herein. For synthesis of polypeptides in this invention the reaction mixture is placed between two flat membranes. The membranes differentiate flows of low molecular weight and high molecular weight components and divide the reactor into three zones: zone for input of feed solution, reaction zone, zone of product removal. The first rather weak flow is formed in the reaction zone. It ensures the reaction mixture movement along the internal part of porous barriers, through which they molecular weight components (including synthesized polypeptides) are removed. The second fast flow is formed in the zone of feed solution input. It ensures penetration of low molecular components via the membrane in the reaction system. The fast flow of low molecular weight components and the slow flow of high molecular components are achieved by creating a tangential flow along the external surface of the first porous barrier and dialysis process for adding feed solution in the zone of synthesis. If high molecular weight components are removed from the reactor, the size of cutoff is chosen from 50 to 100 kD (which is corroborated by example 7).
The speed of penetration of feed solution components to the reactor determined to a greater extent by the dialysis process is insufficient for maintenance of prolonged operation of highly effective cell-free systems.
Requirements to devices for scientific researches and for synthesis of polypeptides in preparative amounts are different. For synthesis of small amounts of polypeptides, from 100 to 200 xcexcg, it is necessary to have a simple and cheap reactor which can ensure synthesis during 20-50 hours without application of expensive equipment and provides an opportunity to choose hand-operated or controlled speeds or flows.
During synthesis of polypeptides in preparative amounts, the device should control the process: operate speeds of flows inside the reactor and provides an opportunity for a prolonged (more than 50 hours) operation at the expense of active mixing or other action protecting from closing of membranes or hollow fibers, provide effective addition of the feed solution and expendable high molecular weight components to the reaction mixture and effective removal of low molecular weight components from the reactor which inhibit the synthesis.
The device (Mozayeny, 1995) is known which operates in the mode of continuous synthesis of peptides and is controlled by computer. The system includes a complicated and expensive equipment (an automatic sampler etc.).
Devices with one reactor from 1.0 ml. (Spirin et al., 1988) to 100.0 ml (Spirin, 1992) are known. Using the principle of dividing the entire reactor volume in several reactors of smaller volume it is possible to apply identical decisions to devices intended for synthesis of polypeptides in laboratory conditions and for prepartive synthesis. In this case routine technology of synthesis of polypeptides in small volumes of the reaction mixture from 50 xcexcl up to 1-5 ml can be used in working with the volume up to 100-200 ml by scaling and increasing the amount of modules operating in parallel.
Devices for maintenance of synthesis in cells (Puchinger et al., 1980; Gebhard et al., 1997; Hu et al., 1997) using the principle of scaling the modules are known. In these devices inputs for supplying a feed solution and outputs for removal of products are connected in parallel for all N reactors. The known devices are designed for maintenance of cell growth and cannot be applied for synthesis of polypeptides, as each of N reaction modules serves to maintain speed, pressure and other parameters of flows of feed solutions and gases necessary for normal functioning of cells.
Known modules for bioreactors on the basis of hollow fibers (Gebhard et al., 1997; Hu et al., 1997) do not take into account the specifity of working with cell-free system. Fibers used in the reactors have the same size of a cutoff and their form reminds a beam placed in a cylinder. Therefore a significant part of the surfaces of hollow fibers contacts each other and reduces the working surface.
The device (Yagihashi et al., 1996) is known whose construction represents two layers of hollow fibers. Each layer consists of glued hollow fibers placed in parallel. Both layers of hollow fibers have the same size of a cutoff and a significant part of their surface is in contact.
The device (Pedersen et al., 1994) is known in which separate modules are single-layer constructions from hollow fibers with the same size of a cutoff.
However this technical decision has been developed for filtration of liquids and cannot be used in reactors for cell-free systems without essential modification in the design since it is intended for working with large volumes of liquid flows.
A large number of designs constructed on the basis of flat membranes is known. They also have disadvantages being intended basically for filtration or dialysis.
The device for synthesis of polypeptides in cell-free system is known (Mozayeny, 1995) whose structure includes two flat membranes. Originally this device (OMEGA TM) was intended for filtration and has a large void volume in the zones of product selection. The feed solution and high molecular weight components are added to the reactor through one input. This device is not intended for assembly in a general construction consisting of N modules.
The prototype of the proposed device for synthesis of polypeptides is the device described in the patent (Alakhov et al., 1985). For synthesis of polypeptides in a mode of product removal the device contains two porous barriers. These barriers can be executed as flat membranes or hollow fibers. The reaction mixture can be placed both from the external and internal sides of hollow fibers.
A disadvantage of the given device is that it contains porous barriers with the same size of a cutoff and provides for removal from the reactor of one flow consisting either of low molecular weight (the cutoff size of 7.5 kD) or high molecular weight (the cutoff size up to 100 kD) fractions.
The presence invention provides a method for synthesis polypeptides in a cell-free system by which products of synthesis are branched in a low molecular weight fraction and a fraction which contains high molecular weight components with the target polypeptide, the main part of the low molecular weight fraction is removed via at least one part of the second porous barrier, the ratio of the volume of the fractions of feed solution and expendable components to the volume of the fraction containing the target polypeptide is chosen, modes of supply of the feed solution and expendable components of the fractions are realized.
It is further object of the present invention to describe a methods for synthesis polypeptides in a cell-free system by which the low molecular weight fraction which consists of removed components including low molecular weight components of the reaction mixture and low molecular weight components of the synthesis is withdrawn from the reaction volume via at least one the second porous barrier only, the mode of supply of fractions of the feed solution and expendable components is realized.
It is therefor, also, an object of present invention to provide the method for obtaining polypeptides in which during the synthesis N cycles are formed, everyone of which consists of at least two steps, at the first step low molecular weight components of the feed solution are supplied to the reactor via the first porous barrier and the low molecular weight fraction with products of synthesis and components of the reaction mixture is removed via the second porous barrier, at the second step the supply and removal channels are switched and low molecular weight components of the feed solution are supplied via the second porous barrier, the low molecular weight fraction or the high molecular weight fraction containing products of synthesis and components of the reaction mixture is removed via the first porous barrier, the mode of supply of of fractions of the feed solution and expendable high molecular weight components is realized.
It is a further object of the present invention to describe a reactor which comprises at least one reactor volume, whose external surface contacts the external surface of the first and second porous barriers, the internal surface of the second porous barrier is connected to the zone of the inlet or outlet of low molecular weight flows, the internal side of the first porous barrier is connected to the zone of the inlet and outlet of low molecular weight flows and flows containing high molecular weight components with the target polypeptide.